- 學位論文
破傷風桿菌焦磷酸水解酵素中色胺酸之功能與螢光分析
Functional and Fluorescence Analyses of Tryptophan Residues in H+-pyrophosphatase of Clostridium tetani
摘要
同型雙次體質子焦磷酸水解酶(H+-PPase; EC 3.6.1.1) 在許多細菌與高等的植物中，藉由水解焦磷酸(PPi)並運送氫質子通過細胞膜，來維持細胞內部pH值的恆定。質子焦磷酸水解酶具有許多基本的功能區，包含PPi結合區、酸性區I及II；這些區域已被證實參與了酵素的催化機制。在本實驗中，破傷風桿菌(Clostridium tetani)質子焦磷酸水解酶中三個原生性的色胺酸(tryptophan)，Trp-75, Trp-365, 和 Trp-602被用來當作內生性的螢光標誌，以觀察其細微局部結構(膜外、細胞膜區及細胞質區)的狀態。藉由與受質類似物(Mg-imidodiphosphate, Mg-IDP)的結合，局部結構的改變使色胺酸能免於受到特定化學修飾物(N-bromosuccinimide, NBS)的修飾破壞，尤其在Trp-602結果最明顯。如果和水解產物Mg-Pi結合，Trp-75 和 Trp-365(但不包含Trp-602)能因結構的改變而不會受到NBS的修飾，此結果也間接說明，Mg-Pi結合時，位於細胞質區的酵素催化區是打開的，同時，不同受質和產物的結合，會使質子焦磷酸水解酶有不同的結構狀態。分析Stern-Volmer relationship 和 Steady-state fluorescence anisotropy指出，位於細胞質區的Trp-602是較暴露、易與外界溶液反應。除此之外，定點突變色胺酸及胰蛋白酶剪切分析指出，Trp-602是非常重要的胺基酸；我們推測該色胺基酸與穩定催化區結構有相關。最後，本實驗中所建立的單一色胺酸突變株，能提供一個新的工具，日後可用來研究質子焦磷酸水解酶的功能與結構分析。
並列摘要
Homodimeric proton-translocating pyrophosphatase (H+-PPase; EC 3.6.1.1) maintains the cytoplasmic pH homeostasis of many bacteria and higher plants by coupling pyrophosphate (PPi) hydrolysis and proton translocation. H+-PPase accommodates several essential motifs involved in the catalytic mechanism, including the PPi binding motif and Acidic I and II motifs. In this study, 3 intrinsic tryptophan residues, Trp-75, Trp-365, and Trp-602, in H+-PPase from Clostridium tetani were used as internal probes to monitor the local conformational states of the periplasm domain, transmembrane region, and cytoplasmic domain, respectively. Upon binding of the substrate analog Mg-imidodiphosphate (Mg-IDP), local structural changes prevented the modification of tryptophan residues by N-bromosuccinimide (NBS), especially at Trp-602. Following Mg-Pi binding, Trp-75 and Trp-365, but not Trp-602, were slightly protected from structural modifications by NBS. These results reveal the conformation of H+-PPase is distinct in the presence of different ligands. Moreover, analyses of the Stern-Volmer relationship and steady-state fluorescence anisotropy also indicate that the local structure around Trp-602 is more exposed to solvent and varied under different environments. In addition, Trp-602 was identified to be an essential residue in the H+-PPase that may potentially be involved in stabilizing the structure of the catalytic region by site-directed mutagenesis and trypsin proteolysis analyses.
並列關鍵字
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延伸閱讀
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